Evaluation of an elastic decellularized tendon-derived scaffold for the vascular tissue engineering application.

Due to the limited success rate of currently available vascular replacements, tissue engineering has received tremendous attention in recent years. A main challenge in the field of regenerative medicine is creating a mechanically functional tissue with a well-organized extracellular matrix, particularly of collagen and elastin. In this study, the native collagen scaffold derived from decellularized tendon sections, as a scaffold having the potential to be used for vascular tissue engineering applications, was studied. We showed that the elasticity of the scaffolds was improved when crosslinked with the bovine elastin. The effect of different concentrations of elastin on mechanical properties of the collagen scaffolds was evaluated of which 15% elastin concentration was selected for further analysis based on the results. Addition of 15% elastin to collagen scaffolds significantly decreased Young's modulus and the tensile stress at the maximum load and increased the tensile strain at the maximum load of the constructs as compared to those of the collagen scaffolds or control samples. Moreover, tubular elastin modified collagen scaffolds showed significantly higher burst pressure compared to the control samples. Smooth muscle cells and endothelial cells cultured on the elastin modified collagen scaffolds showed high viability (>80%) after 1, 3, and 7 days. Furthermore, the cells showed a high tendency to align with the collagen fibers within the scaffold and produced their own extracellular matrix over time. In conclusion, the results show that the decellularized tendon sections have a great potential to be used as scaffolds for vascular tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1225-1234, 2019.

Nanostructured Tendon-Derived Scaffolds for Enhanced Bone Regeneration by Human Adipose-Derived Stem Cells.

Decellularized matrix-based scaffolds can induce enhanced tissue regeneration due to their biochemical, biophysical, and mechanical similarity to native tissues. In this study, we report a nanostructured decellularized tendon scaffold with aligned, nanofibrous structures to enhance osteogenic differentiation and in vivo bone formation of human adipose-derived stem cells (hADSCs). Using a bioskiving method, we prepared decellularized tendon scaffolds from tissue slices of bovine Achilles and neck tendons with or without fixation, and investigated the effects on physical and mechanical properties of decellularized tendon scaffolds, based on the types and concentrations of cross-linking agents. In general, we found that decellularized tendon scaffolds without fixative treatments were more effective in inducing osteogenic differentiation and mineralization of hADSCs in vitro. When non-cross-linked decellularized tendon scaffolds were applied together with hydroxyapatite for hADSC transplantation in critical-sized bone defects, they promoted bone-specific collagen deposition and mineralized bone formation 4 and 8 weeks after hADSC transplantation, compared to conventional collagen type I scaffolds. Interestingly, stacking of decellularized tendon scaffolds cultured with osteogenically committed hADSCs and those containing human cord blood-derived endothelial progenitor cells (hEPCs) induced vascularized bone regeneration in the defects 8 weeks after transplantation. Our study suggests that biomimetic nanostructured scaffolds made of decellularized tissue matrices can serve as functional tissue-engineering scaffolds for enhanced osteogenesis of stem cells.

Biocompatibility and degradation of tendon-derived scaffolds.

Decellularized extracellular matrix has often been used as a biomaterial for tissue engineering applications. Its function, once implanted can be crucial to determining whether a tissue engineered construct will be successful, both in terms of how the material breaks down, and how the body reacts to the material's presence in the first place. Collagen is one of the primary components of extracellular matrix and has been used for a number of biomedical applications. Scaffolds comprised of highly aligned collagen fibrils can be fabricated directly from decellularized tendon using a slicing, stacking, and rolling technique, to create two- and three-dimensional constructs. Here, the degradation characteristics of the material are evaluated in vitro, showing that chemical crosslinking can reduce degradation while maintaining fiber structure. In vivo, non-crosslinked and crosslinked samples are implanted, and their biological response and degradation evaluated through histological sectioning, trichrome staining, and immunohistochemical staining for macrophages. Non-crosslinked samples are rapidly degraded and lose fiber morphology while crosslinked samples retain both macroscopic structure as well as fiber orientation. The cellular response of both materials is also investigated. The in vivo response demonstrates that the decellularized tendon material is biocompatible, biodegradable and can be crosslinked to maintain surface features for extended periods of time in vivo. This study provides material characteristics for the use of decellularized tendon as biomaterial for tissue engineering.

A toolkit of thread-based microfluidics, sensors, and electronics for 3D tissue embedding for medical diagnostics.

Threads, traditionally used in the apparel industry, have recently emerged as a promising material for the creation of tissue constructs and biomedical implants for organ replacement and repair. The wicking property and flexibility of threads also make them promising candidates for the creation of three-dimensional (3D) microfluidic circuits. In this paper, we report on thread-based microfluidic networks that interface intimately with biological tissues in three dimensions. We have also developed a suite of physical and chemical sensors integrated with microfluidic networks to monitor physiochemical tissue properties, all made from thread, for direct integration with tissues toward the realization of a thread-based diagnostic device (TDD) platform. The physical and chemical sensors are fabricated from nanomaterial-infused conductive threads and are connected to electronic circuitry using thread-based flexible interconnects for readout, signal conditioning, and wireless transmission. To demonstrate the suite of integrated sensors, we utilized TDD platforms to measure strain, as well as gastric and subcutaneous pH in vitro and in vivo.

In Vivo Peripheral Nerve Repair Using Tendon-Derived Nerve Guidance Conduits.

There is an urgent need for a peripheral nerve repair product that can match or exceed the abilities of the current "gold-standard", nerve autografts. Using a sectioning-based fabrication technique, decellularized tendon sections formed into tubular conduits that maintain the native structure of the collagen. Our previous studies have demonstrated that these collagen structures provide nanotopographical growth guidance cues for regenerating neurons and support glia. Here, the regenerative abilities of the tendon-derived nerve guidance conduits to repair a critically sized defect (15 mm) are evaluated in a rat sciatic nerve model. Using the conduits, functional recovery occurs at a similar rate to isografts, when evaluated with a sciatic function index test. However, muscular recovery, as measured by gastrocnemius weight, was not as great in the conduit-treated group. Both conduit and isograft repairs are histologically evaluated using Masson's trichrome stain and immunofluorescent staining for neurofilament-160 and S100 (markers for neurons and Schwann cells, respectively). This evaluation shows that by week 14, conduits promote regrowth of both neuronal tissue and some physiological support structures, such as blood vessels and epi/perineurium-like structures. Lastly, positive staining for these two markers at week 14 is calculated as a quantitative means of assessment, and shows greater total content of neurofilament-160 and S100 in conduits than in isografts, but a smaller percent area, which may be a result of the greater cross-sectional area of the conduit.

Laminar Tendon Composites with Enhanced Mechanical Properties.

PURPOSE: A strong isotropic material that is both biocompatible and biodegradable is desired for many biomedical applications, including rotator cuff repair, tendon and ligament repair, vascular grafting, among others. Recently, we developed a technique, called "bioskiving" to create novel 2D and 3D constructs from decellularized tendon, using a combination of mechanical sectioning, and layered stacking and rolling. The unidirectionally aligned collagen nanofibers (derived from sections of decellularized tendon) offer good mechanical properties to the constructs compared with those fabricated from reconstituted collagen. METHODS: In this paper, we studied the effect that several variables have on the mechanical properties of structures fabricated from tendon slices, including crosslinking density and the orientation in which the fibers are stacked. RESULTS: We observed that following stacking and crosslinking, the strength of the constructs is significantly improved, with crosslinked sections having an ultimate tens ile strength over 20 times greater than non-crosslinked samples, and a modulus nearly 50 times higher. The mechanism of the mechanical failure mode of the tendon constructs with or without crosslinking was also investigated. CONCLUSIONS: The strength and fiber organization, combined with the ability to introduce transversely isotropic mechanical properties makes the laminar tendon composites a biocompatiable material that may find future use in a number of biomedical and tissue engineering applications.

Pericellular hydrogel/nanonets inhibit cancer cells.

Fibrils formed by proteins are vital components for cells. However, selective formation of xenogenous nanofibrils of small molecules on mammalian cells has yet to be observed. Here we report an unexpected observation of hydrogel/nanonets of a small D-peptide derivative in pericellular space. Surface and secretory phosphatases dephosphorylate a precursor of a hydrogelator to trigger the self-assembly of the hydrogelator and to result in pericellular hydrogel/nanonets selectively around the cancer cells that overexpress phosphatases. Cell-based assays confirm that the pericellular hydrogel/nanonets block cellular mass exchange to induce apoptosis of cancer cells, including multidrug-resistance (MDR) cancer cells, MES-SA/Dx5. Pericellular hydrogel/nanonets of small molecules to exhibit distinct functions illustrates a fundamentally new way to engineer molecular assemblies spatiotemporally in cellular microenvironment for inhibiting cancer cell growth and even metastasis.

Enhanced intracellular siRNA delivery using bioreducible lipid-like nanoparticles.

A new library of lipid-like nanoparticles (lipidoids) comprising disulfide bond is developed for siRNA delivery. Bioreducible lipidoids deliver siRNA with greater efficiency than nonbioreducible lipidoids with similar chemical structures. A siRNA release investigation, as well as an intracellular siRNA trafficking study, reveals that the degradation of bioreducible lipidoid in a strongly reductive intracellular environment boosts siRNA release and enhances siRNA gene knockdown efficiency.

Combinatorially designed lipid-like nanoparticles for intracellular delivery of cytotoxic protein for cancer therapy.

An efficient and safe method to deliver active proteins into the cytosol of targeted cells is highly desirable to advance protein-based therapeutics. A novel protein delivery platform has been created by combinatorial design of cationic lipid-like materials (termed "lipidoids"), coupled with a reversible chemical protein engineering approach. Using ribonuclease A (RNase A) and saporin as two representative cytotoxic proteins, the combinatorial lipidoids efficiently deliver proteins into cancer cells and inhibit cell proliferation. A study of the structure-function relationship reveals that the electrostatic and hydrophobic interactions between the lipidoids and the protein play a vital role in the formation of protein-lipidoid nanocomplexes and intracellular delivery. A representative lipidoid (EC16-1) protein nanoparticle formulation inhibits cell proliferation in vitro and suppresses tumor growth in a murine breast cancer model.

The behavior of neuronal cells on tendon-derived collagen sheets as potential substrates for nerve regeneration.

Peripheral nervous system injuries result in a decreased quality of life, and generally require surgical intervention for repair. Currently, the gold standard of nerve autografting, based on the use of host tissue such as sensory nerves is suboptimal as it results in donor-site loss of function and requires a secondary surgery. Nerve guidance conduits fabricated from natural polymers such as collagen are a common alternative to bridge nerve defects. In the present work, tendon sections derived through a process named bioskiving were studied for their potential for use as a substrate to fabricate nerve guidance conduits. We show that cells such as rat Schwann cells adhere, proliferate, and align along the fibrous tendon substrate which has been shown to result in a more mature phenotype. Additionally we demonstrate that chick dorsal root ganglia explants cultured on the tendon grow to similar lengths compared to dorsal root ganglia cultured on collagen gels, but also grow in a more oriented manner on the tendon sections. These results show that tendon sections produced through bioskiving can support directional nerve growth and may be of use as a substrate for the fabrication of nerve guidance conduits.

DOPE facilitates quaternized lipidoids (QLDs) for in vitro DNA delivery.

This paper describes the synthesis of a combinatorial library of quaternized lipidoids (QLDs) and an evaluation of their abilities to facilitate in vitro DNA delivery. The QLDs alone showed low efficiency for DNA delivery. By formulating liposomes with a neutral helper lipid, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the capability of QLDs for gene transfection is significantly enhanced due to the fusogenic properties of DOPE which facilitate endosomal escape and cargo delivery. We further optimized the liposome composition and DNA dose for gene transfection and investigated the structure-activity relationships of the lipidoid library in DNA delivery. FROM THE CLINICAL EDITOR: This paper describes the synthesis and evaluation of a combinatorial library of quaternized lipidoids to facilitate in vitro DNA delivery, which occurs at a low level but can be enhanced with DOPE. The authors also further optimized the liposome composition and DNA dose for delivery and investigated the structure-activity relationships of the lipidoid library.

Slicing, stacking and rolling: fabrication of nanostructured collagen constructs from tendon sections.

A novel method for fabricating both multilayer stacked 2D and 3D tubular constructs composed of sheets of aligned collagen fibers is described. These structures are created by decellularizing native tendon and sectioning the material into thin sheets using a cryo-microtome. This fabrication method preserves the collagens natural strength as well as the fiber structure which would aid in directing aligned cell growth.

A combinatorial library of unsaturated lipidoids for efficient intracellular gene delivery.

A combinatorial library of unsaturated lipidoids was synthesized through the Michael addition of amines to oleyl acrylamide. Their capability in facilitating in vitro gene delivery was evaluated by transfecting HeLa cells with EGFP-encoding plasmid DNA and mRNA. The preliminary screening results indicated that lipidoids with unsaturated oleyl tails are superior transfection agents compared to saturated lipidoids with n-octadecyl tails under the same conditions. The different transfection abilities of the unsaturated and saturated lipidioids were ascribed to the large, tightly packed lipoplexes of saturated lipidoids. The potential applications of the library of lipidoids were further expanded by looking at their ability to transfect fibroblasts as well as different cancerous cell lines.